Nicotinic acid adenine dinucleotide phosphate (NAADP) as a novel and potent intracellular Ca2+ signaling second messenger has been reported to mobilize Ca2+ from an acidic Ca2+ store or lysosomes to trigger a global Ca2+ response. However, it remains unknown how this signaling nucleotide activates Ca2+ release from lysosomes and what is the identity of NAADP-sensitive Ca2+ release channels on this organelle. The present proposal will test a central hypothesis that an NAADP-sensitive Ca2+ release channel in lysosomes of coronary arterial smooth muscle cells (CASMCs) is characteristic of transient receptor potential-mucolipin1 (TRP-ML1), which mediates local Ca2+ bursts from lysosomes and leads to a two-phase Ca2+ release participating in the vasomotor response of coronary arteries to agonists. To test this hypothesis, four Specific Aims are proposed. Specific Aim 1 will characterize lysosomal Ca2+ release channels from CASMCs by lipid bilayer channel reconstitution technique and then explore the mechanisms regulating these channels related to Ca2+ or H+ sensitivity. Specific Aim 2 attempts to demonstrate the identity of NAADP-sensitive Ca2+ release channels to be TRP-ML1 in purified lysosomes and in intact coronary arterial myocytes using different approaches including biochemical and molecular detections, electrophysiological approaches, confocal microscopy, FRET, and use of TRP-ML1 deficient cells. In Specific Aim 3, we will identify the structural and functional junction between the lysosomes and SR in CASMCs using electron microscopy and to explore the mechanisms by which two organelles interact via this junction by using total internal reflectance microscopy. Finally, Specific Aim 4 will determine whether TRP-ML1 as a NAADP-sensitive Ca2+ release channel contributes to the regulation of vascular tone and vasomotor response by video microscopy of isolated pressurized small coronary arteries. These proposed studies will, for the first time, link NAADP-induced lysosomal Ca2+ release to TRP-ML1 channels and thereby reveal a novel function of lysosomal TRP- ML1 in the regulation of intracellular Ca2+ levels in vascular smooth muscle cells. The findings will significantly increase our understanding of an important spatiotemporal regulation of intracellular Ca2+ signaling associated with the lysosomal Ca2+ store or two- pool mechanism PUBLIC HEALTH RELEVANCE: This grant proposal seeks to study a novel signaling mechanism by which calcium concentrations within muscle cells of the heart artery wall are regulated. Although there are some reports indicating that calcium within these cells are released to produce contraction of the arteries through a two-phase release process, it is unknown how this two-phase release happens. The present grant proposal will demonstrate that a protein called as transient receptor potential-mucolipin1 serves as a trigger to mediate this two- phase calcium release within the cells. The findings will importantly contribute to our understanding of the mechanisms responsible for contraction of the heart arteries and therefore may help develop new therapy for coronary arterial disease to prevent heart attack in the future.